Project Summary Luciferase Bioluminescence Imaging (BLI) is a non-invasive method for monitoring diverse processes in living systems. BLI couples in vitro and in vivo biological events to the bioluminescent output of the enzyme luciferase. The potential detection limit, versatility, and temporal and spatial resolution of BLI assays can even exceed fluorescence-based methods when in the context of deep tissue or whole animal imaging applications. For these reasons, BLI has been a foundational imaging tool in whole animal imaging applied to both the study of diseases as well as in the evaluation of potential therapies. The most frequently used enzyme for BLI is Firefly luciferase (F-Luc), the luciferase enzyme from the North American firefly (Photinus pyralis); engineering of F-Luc is needed to expand and improve its applications. Here, we propose methodological advances to F-Luc engineering, which should yield important biochemical data, generate potentially useful enzymes, and provide important precedent for further studies in the field. One of the current challenges of F-Luc engineering is the dearth of biochemical data that can be used to guide evolution efforts in the laboratory. In Aim 1, we propose to use high-throughput biochemical approaches, which combine high- throughput screening, next generation sequencing, and computational analysis to measure four important biochemical parameters (brightness, emission color, thermostability, and substrate selectivity) for 500 amino acid mutations en masse. This work will provide insight into the relationship between sequence and protein function for a substantial portion of the luciferase protein, which will inform future engineering efforts by our lab and others, as well as lead to a broader understanding of luciferase function. A second major challenge in F-Luc engineering is that improvement of one property (e.g. emission color) is often coupled to loss of the enzyme?s brightness, diminishing the value of engineered F-Luc mutants. In Aim 2, we propose to apply a method that we have recently developed to identify brighter luciferase enzymes; we will apply the method to two recently discovered F-Luc mutants, which have applications in multi-component imaging applications. In addition to improved enzymes with immediate imaging applications, this work will constitute the first direct screen for luciferase brightness to date, providing a methodological advancement for the field of luciferase engineering.